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Abstract

Prostate cancer (PCa) is one of the most common malignancies among men and is the second leading cause of cancer‑associated mortality in the developed world. Androgen deprivation therapy (ADT) is the most common treatment for PCa. However, the majority of androgen‑sensitive PCa patients will eventually develop resistance to ADT and the disease will become androgen‑independent. There is, therefore, an immediate requirement to develop effective therapeutic techniques towards the treatment of recurrent PCa. Oxibendazole (OBZ) is an anthelmintic drug that has also shown promise in the treatment of malignancies. In the present study, the capability of OBZ to repress the growth of PCa cells was assessed in human androgen‑independent PCa 22Rv1 and PC‑3 cell lines. The growth of the 22Rv1 and PC‑3 cell lines, as assessed with a trypan blue exclusion assay, was markedly inhibited by OBZ treatment in vitro, with half‑maximal inhibitory concentration values of 0.25 and 0.64 µM, respectively. The mean size of 22Rv1 tumors in nude mice treated with OBZ (25 mg/kg/day) was 47.96% smaller than that of the control mice. Treatment with OBZ increased the expression of microRNA‑204 (miR‑204), as determined by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and the level of p53 as determined with western blotting, two well‑characterized tumor suppressor genes. When miR‑204 expression was knocked down by introduction of an miR‑204 inhibitor, the inhibitory effect of OBZ was markedly reduced; however, when it was overexpressed, the inhibitory efficiency of OBZ was markedly higher, indicating that upregulation of miR‑204 is key for the efficacy of OBZ. Additionally, OBZ was demonstrated with RT‑qPCR to repress the expression of the androgen receptor, and by western blotting to reduce prostate‑specific androgen in 22Rv1 cells. The results suggest that OBZ has potential for clinical use in the treatment of recurrent PCa.

Introduction

Prostate cancer (PCa) is the most common malignancy
of the male genitourinary system and primarily occurs in aging men
(1). PCa is the second leading cause
of cancer-associated mortality in developed countries (2). Conventional treatments for PCa include
surgery, chemotherapy, hormonal therapy, radiation therapy,
radiofrequency ablation, high-intensity focused ultrasound and
cryosurgery (3–5). Currently, androgen-deprivation therapy
(ADT) is widely used for PCa treatment; however, the majority of
PCa patients relapse within 1 to 3 years and develop
androgen-independent disease, which is unresponsive to ADT
(6–8).
At present, there is no effective therapy for recurrent PCa
(9). Therefore, a novel therapeutic
method for PCa is required. Recently, the US Food and Drugs
Administration approved novel compounds for the treatment of PCa,
including abiraterone acetate (10),
enzalutamide (11), sipuleucel-T
(12), and Alpharadin (radium-233)
(13). One study proposed that
certain non-antitumor chemicals may have an antitumor effect
(14). Sulfur, which is widely used
for detoxifying the body and the treatment of scabies in
traditional Chinese medicine (15,16), has
been shown to suppress the growth of PCa in vivo (17). Itraconzole, a common triazole
antifungal drug in widespread clinical use, has shown evidence of
clinical anticancer effects, including against PCa (18).

Oxibendazole
(methyl-5-n-propoxy-2-benzimidazole-carbamate; OBZ), was first
synthesized in 1973 (19). OBZ is a
benzimidazole drug that is used to treat infection by roundworms,
strongyles, pinworms, threadworms and lungworm infestation in
horses and other animals (20–22). The
benzimidazole derivatives exhibit, among others, anti-ulcerative
(23), anti-inflammatory (24), antibacterial (25), and anti-carcinogenic (26) bioactivities. These drugs are widely
available for veterinary use and a number of them, such as
thiabendazole, albendazole and mebendazole, have been used in human
medicine for several years (27). A
previous study found that benzimidazoles could have potential
cytostatic effects, through the inhibition of microtubule formation
and glucose uptake (28). Notably,
albendazole, mebendazole and flubendazole, which are all
benzimidazole drugs, have been observed to inhibit tumor growth
(29).

The ability of OBZ to suppress growth in PCa was
assessed in the present study using the PCa 22Rv1 and PC-3 cell
lines. Previous studies have shown that growth of recurrent PCa
cells (also termed androgen-independent PCa cells) depends on the
androgen receptor (AR) or the AR signaling pathway (ASP), although
it is independent of androgens themselves (30,31). The
22Rv1 cell line is an androgen-independent PCa epithelial cell line
that is representative of clinical recurrent PCa (32). This cell line expresses AR and
prostate-specific antigen (PSA) (33,34), which
has been widely used as a clinically diagnostic biomarker and a key
prognostic factor for PCa (35).
However, the androgen-independent PC-3 cells do not express AR or
PSA (36). The two markers are
frequently used to evaluate the anti-PCa effects of diverse
chemicals (37–39).

In the present study, 22Rv1 cells were studied in
vitro and in vivo, and PC-3 cells were studied in
vitro. The aim of the study was to investigate the inhibitory
effect of oxibendazole on prostate cancer cells.

Materials and methods

Drugs and animals

OBZ was purchased from Selleck Chemicals, Inc.
(Houston, TX, USA) and was prepared as homogeneous suspensions in
corn oil and administered to the nude mice by gavage. A total of 20
specific pathogen-free (SPF) male BALB/c nude mice aged between 6
and 8 weeks (weight range, 18–25 g) were purchased from Shanghai
SLAC Laboratory Animal Co., Ltd. (Shanghai, China), maintained
under SPF conditions with a 12 h light-dark cycle and provided with
food and water ad libitum. Mice in the experiment were
randomly divided into two groups, the control and the OBZ-treated
groups, with 10 mice in each group. Ethical approval for the
present study was obtained from the Animal Ethics Committee of
Shanghai Institute of Planned Parenthood Research (Shanghai,
China).

Cell proliferation assays

Cells were seeded in 96-well plates at a cell
density of 1×104 cells per well in 100 µl RPMI-1640
medium containing 10% fetal bovine serum and 1%
penicillin-streptomycin, and incubated at 37°C with an atmosphere
of 5% CO2 overnight. 22Rv1 and PC-3 cells were treated
with 0.12, 0.25, 0.50, 1.00, 2.00 and 3.00 µM OBZ for 48 h, or with
0.25 and 1.00 µM OBZ for 96 h. In order to assess the role of
miR-204 in mediating the effect of OBZ, 22Rv1 and PC-3 cells were
transfected with the miR-204 or miR-204 inhibitor, followed by
treatment with 1 µM OBZ or dimethyl sulfoxide (DMSO; as a control)
for 48 h. Cells were trypsinized and live cell numbers were counted
in four areas under an inverted microscope (magnification, ×40)
using a hemocytometer and the trypan blue exclusion assay.

Flow cytometry

Apoptosis was determined using a double-staining
Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit
(BD Biosciences Inc., Franklin Lakes, NJ, USA). The 22Rv1 and PC-3
cells were treated with DMSO control or 0.25 µM OBZ. After 48 h,
the cells were collected, washed in phosphate-buffered saline (PBS)
and suspended in binding buffer. The cells were then stained using
annexin V and propidium iodide (PI) (5 µl). Following incubation
for 15 min at room temperature in the dark, the cells were diluted
and analyzed using a flow cytometer (Beckman Coulter, Inc.,
Atlanta, GA, USA). When green fluorescence (FITC) was plotted
against red fluorescence (PI), the cell populations could be
detected in a dot-plot that indicated the following conditions:
Viable cells (FITC−/PI−), early apoptotic
cells (FITC+/PI−) and late apoptotic cells
(FITC+/PI+). The data were reported as the
percentage of early apoptotic cells
(FITC+/PI−) and late apoptotic cells
(FITC+/PI+).

Xenograft tumor development in nude
mice

At the exponential growth stage, 22Rv1 cells were
harvested, washed and suspended in PBS. A trypan blue exclusion
assay was performed to ensure cell viability (>99%) prior to
inoculation. The cells were counted and 2×106 cells
suspended in 0.1 ml PBS were subcutaneously injected into the right
flank of each mouse. At 10 days after tumor cell inoculation, each
mouse in the OBZ-treated group was provided with 25 mg/kg
homogeneous suspension of OBZ by intragastric gavage. The treatment
was administered once a day for 14 days; mice in the control group
was provided with the same amount of corn oil. Tumor size was
measured in two dimensions every other day. Tumor volume (measured
in cm3) was calculated using the following formula:
Tumor volume=axb2×0.5 (a, length; b, width).

Total RNA was extracted using TRIzol reagent
(Invitrogen; Thermo Fisher Scientific, Inc.) according to the
manufacturer's instructions. First-strand cDNA synthesis was
performed on the total RNAs using Quant Reverse Transcriptase
(Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's
instructions with a PCR machine (cat. no. 580BR6819; RiboBio Co.,
Ltd, Guangzhou, China). Gene amplifications were performed with a
Eco Real time PCR System (Model Ec-100-1001; Illumina, Inc., San
Diego, CA, USA) using SYBR-Green (Thermo Fisher Scientific, Inc.).
Gene expression in cells and tumors was measured using qPCR. The
cycling conditions of GAPDH, AR, ARN1 and CD44 were as followed:
95°C for 3 min, 95°C for 20 sec and 58°C for 20 sec, for 40 cycles.
The cycling conditions of U6, miR-204 and miR-34a were as follows:
95°C for 1 min, 95°C for 15 sec and 60°C for 15 sec, for 40 cycles.
Bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of
qPCR primers for each set) specific for miR-34a were designed by
RiboBio. Primers for β-actin, AR and 5′-3′ exoribonuclease 1 (XRN1)
were purchased from Sangon Biotech, Inc. (Shanghai, China). The
sequences of the primers are included in Table I. The relative expression of genes was
calculated using the 2−ΔΔCq method (40). The mean Cq was calculated from
triplicate PCRs.

Statistical analysis

Data were analyzed using SPSS software, version 11.5
(SPSS, Inc., Chicago, IL, USA). Differences in the continuous
variable ‘tumor volume’ were compared using a one-way analysis of
variance followed by a Student-Newman-Keuls test. Densitometry
analysis of western blots was performed using Image J software
(version 1.37, National Institute of Health, Bethesda, MD, USA).
Data are presented as the mean ± standard error of the mean.
P<0.05 was considered to indicate a statistically significant
difference.

Results

OBZ inhibits growth of 22Rv1 and PC-3
cells

The ability of OBZ to inhibit the growth of 22Rv1
and PC-3 cells was determined by counting cell number. OBZ markedly
inhibited the cell viability of 22Rv1 and PC-3 cells in a
dose-dependent manner (Fig. 1A). As
little as 0.12 µM of OBZ was observed to significantly inhibit the
growth of the 22Rv1 and PC-3 cells, respectively (both P<0.05).
The 22Rv1 cells were more sensitive to OBZ treatment, with a
half-maximal inhibitory concentration (IC50) value of
0.25 µM, compared with 0.64 µM in PC-3 cells. OBZ inhibited the
cell viability of 22Rv1 and PC-3 cells in a time-dependent manner
(Fig. 1B and C). These results
demonstrated that OBZ inhibited the growth of PCa cells in
vitro with varied efficiency.

OBZ causes apoptosis of 22Rv1 and PC-3
cells

The apoptosis-inducing capability of OBZ in 22Rv1
and PC-3 cells was evaluated by Annexin V-FITC and PI double
staining. Provided that the IC50 value was 0.25 µM in
22Rv1 cells, 0.25 µM OBZ was used to treat 22Rv1 and PC-3 cells for
48 h. A notable increase in the number of apoptotic cells was
observed in the OBZ-treated group compared with DMSO-treated cells
(the negative control) (Fig. 2A and
B). The apoptotic rate of 22Rv1 cells was 1.41% in DMSO-treated
cells and 9.45% in OBZ-treated cells. The apoptotic rate in PC-3
cells was 0.92 and 4.58% in DMSO- and OBZ-treated cells,
respectively (Fig. 2C). These results
indicated that treatment with OBZ resulted in an increased
apoptotic rate in PCa cells, and the apoptosis-inducing capability
of OBZ was more marked in 22Rv1 compared with PC-3 cells.

To investigate the mechanism of the inhibitory
effect of OBZ in PCa cells, the effect of OBZ on the expression of
p53 and p21 (41,42), which are known to be the key
regulators of apoptosis, was measured via western blot analysis in
the OBZ-treated cells. An OBZ dose of ≥0.25 µM markedly increased
p53 and p21 expression in 22Rv1 cells (Fig. 2D). These results suggested that
upregulation of p53 and p21 served an important role in the
OBZ-induced apoptosis of 22Rv1 cells.

OBZ inhibits 22Rv1 tumor growth in
nude mice

The antitumor effect of OBZ was next evaluated in
vivo. First, 22Rv1 cells were injected into the right flank of
nude mice. Approximately 10 days after injection of the cells, the
tumor sizes were measurable. On day 10, the mice were treated with
OBZ (25 mg/kg) by intragastric gavage. The treatment was
administered once a day for 14 days. The control group of mice was
treated in the same way, but OBX was substituted with corn oil. OBZ
significantly repressed tumor growth in a time-dependent manner,
with a significant difference identified at 20 days after cancer
cell inoculation (P<0.05; Fig.
3A). The mean tumor volume of the OBZ-treated group was 0.63
cm3, whereas in the control group it was 1.20
cm3; OBZ inhibited growth of the tumor by ~47.96%.
Additionally, the mean body weight of the tumor-bearing mice was
24.06±1.28 and 23.10±3.39 g in the OBZ-treated and control groups,
respectively. However, this difference was not statistically
significant, demonstrating that OBZ did not exert a significant
general toxic effect in vivo, consistent with the results of
a previous study (43).

The mechanism for the tumor-suppressive effect of
OBZ in vivo was also studied. First, the expression of p53
and p21 in the tumor tissues of OBZ-treated mice and their negative
control was measured using western blot analysis. The density of
the two proteins increased by 821.34 and 75.18%, respectively, in
the OBZ-treated tumor compared with the control samples (Fig. 3B). Therefore, the in vivo
results are consistent with those observed in vitro.

Given that PSA is a clinically diagnostic biomarker
and key prognostic factor for PCa (35), anti-PCa chemicals have also been
assessed for their effect on reducing the level of PSA either in
PCa cells or in the blood (44). The
level of PSA in OBZ-treated 22Rv1 tumors was measured via western
blotting in the present study. The concentration of PSA decreased
by 78.16% more in OBZ-treated tumors compared with the control,
indicating that OBZ repressed PSA expression.

The AR serves a critical role in the progression and
recurrence of PCa (45), and is also
the transcriptional activator of PSA expression (46). Therefore, downregulation of PSA by OBZ
indicates that OBZ affects the AR or ASP. Recently, a study
reported the existence of the AR-miR-204-XRN1 axis in PCa cells,
which has a dual regulatory function in mediating the growth of
different PCa cells (47). In this
axis, androgen/AR raises the level of XRN1, a target of miR-204, by
inhibiting expression of miR-204. Accordingly, whether OBZ affects
the AR-miR-204-XRN1 axis in 22Rv1 tumors was investigated. Levels
of AR, miR-204 and XRN1 in the mouse tumors were measured by qPCR.
Expression of AR and XRN1 was significantly lower in OBZ-treated
tumors than in the control (both P<0.01; Fig. 3C). By contrast, OBZ raised the
expression of miR-204 ~115% (Fig.
3D). Together, these results indicated that OBZ interfered with
the AR-miR-204-XRN1 axis in PCa tumors.

miR-34a has been reported to be a tumor suppressive
miRNA (48). miR-34a represses the
growth of PCa cells by targeting the AR (49,50). As
such, the level of miR-34a in OBZ-treated 22Rv1 tumors was measured
in the present study. OBZ treatment raised the expression of
miR-34a by ~5-fold (Fig. 3D). These
results, therefore, were consistent with the hypothesis that OBZ
suppresses AR expression by raising the level of miR-34a in 22Rv1
cells.

Manipulated expression of miR-204
affects the sensitivity of 22Rv1 cells to OBZ

To evaluate the role of a disturbed AR-miR-204-XRN1
axis in the inhibition of growth of 22Rv1 tumors using OBZ, the
effect of OBZ on miR-204 expression was studied in cultured 22Rv1
cells and PC-3 cells. miR-204 has been widely shown to be a tumor
suppressive gene in multiple types of cancer, including
AR-expressing PCa cells (47,51–53).
However, miR-204 has been reported to be an oncomiR in the PC-3
cell line (47), which is AR-negative
and represents a neuroendocrine-like PCa (NEPC) cell (54). The present results, based on RT-qPCR
data, showed that 1.00 µM OBZ significantly raised the expression
of miR-34a and miR-204 in 22Rv1 cells by 197.07 and 185.11%,
respectively (both P<0.05; Fig.
4A). Additionally, 1.00 µM OBZ significantly raised the
expression of miR-34a and miR-204 in PC-3 cells by 44.77 and
87.46%, respectively (both P<0.05; Fig. 4B).

Cluster of differentiation 44 (CD44) is a PCa
stem-cell marker that is expressed in NEPC cells, including PC-3
cells, but not in AR-expressing PCa cells (54–56). A
previous report demonstrated that CD44 is the target gene of
miR-34a (57). Considering that OBZ
raised the expression of miR-34a in PC-3 cells in the present
study, it was speculated that OBZ may suppress the expression of
CD44. Notably, 0.25 µM OBZ significantly reduced the expression of
CD44 compared with the control (by 60.22%; P<0.01). CD44
expression was almost completely repressed in PC-3 cells that were
treated with 1.00 µM OBZ (Fig. 4C).
These results supported the hypothesis that OBZ represses CD44
expression by raising the level of miR-34a in PC-3 cells.

If the upregulation of miR-204 is essential for the
growth inhibition mediated by OBZ, then ectopic expression of
miR-204 should enhance its inhibitory efficiency. To test the
hypothesis, 22Rv1 cells and PC-3 cells were stably infected with a
recombinant lentivirus expressing miR-204 and green fluorescent
protein (GFP). As a control, the cells were stably infected with a
recombinant lentivirus expressing only GFP. Overexpression of
miR-204 significantly raised the inhibitory effect of OBZ (by
22.77%; P<0.05; Fig. 4D),
suggesting that miR-204 is important to the tumor-suppressive
effect of OBZ in 22Rv1 cells. However, the ectopic expression of
miR-204 did not significantly change the effect of OBZ in PC-3
cells (P=0.1595; Fig. 4E). To exclude
further any potential artificial effect exerted by the
overexpression of miR-204 in 22Rv1 cells, the study also assessed
whether knockdown of miR-204 could change effect of OBZ. 22Rv1
cells were transiently transfected with a miR-204 inhibitor or a
non-targeting control prior to treatment with OBZ. OBZ exerted its
inhibitory effect with a lower (by 36.11%) efficiency in cells that
were transfected with the miR-204 inhibitor than in those
transfected with the control (Fig.
4F). Taken together, these results indicated that upregulation
of miR-204 is key for the tumor-suppressive effect of OBZ in 22Rv1
cells.

Discussion

The present study demonstrated that OBZ markedly
inhibited the growth of androgen-independent PCa cells in cultured
cells and xenograft models (Figs. 1
and 3), at least partially by
inducing the apoptosis of the PCa cells (Fig. 2).

AR serves a key role in PCa progression (45), and is the target of drugs used in PCa
therapy. AR-regulated genes have been extensively studied in
primary and recurrent PCa, but the key genes under the control of
AR have remained elusive. Activation of the AR in the PCa
AR/miR-204/XRN1/miR-34a positive feedback loop (47) upregulates XRN1 expression by
repressing miR-204 expression, whereas XRN1 selectively degrades
miR-34a, eventually resulting in the raised expression of AR, since
AR is a target gene of miR-34a.

The regulation of the AR/miR-204/XRN1/miR-34a
positive feedback loop is severely disturbed in OBZ-treated 22Rv1
tumors, given that OBZ upregulated the expression of miR-204 and
miR-34a, but inhibited the expression of AR and XRN1 in the present
study (Fig. 3B and C). miR-204 has
been reported to act as a tumor suppressor, and is markedly
downregulated in various types of solid malignant tumors (51–53) and
AR-positive PCa tumors (47). The
tumor suppressor activity of miR-204 is mediated not only by its
capability to reduce apoptosis and inhibit the
epithelial-to-mesenchymal transition (EMT), but also by its power
to increase the efficiency of chemotherapy of cancer (58–60). For
example, overexpression of miR-204 increased the responsiveness of
gastric cancer cells to 5-fluorouracil and oxaliplatin treatment in
gastric cancer (52). Similarly,
ectopic expression of miR-204 markedly raised the sensitivity of
22Rv1 cells to OBZ in the present study (Fig. 4D). Consistent with these findings,
knockdown of miR-204 promoted OBZ resistance (Fig. 4F). These results, together with the
observation that OBZ treatment upregulated miR-204, indicates that
miR-204 serves a key role in mediating the anticancer effect of
OBZ.

Besides interfering with the AR/miR-204/XRN1/miR-34a
regulatory loop, OBZ also upregulated the expression of p53 and
p21. The upstream event induced by OBZ in 22Rv1 cells is currently
unknown. It has been noted that the effect of OBZ on the expression
of miR-34a is markedly stronger in 22Rv1 cells, which express
wild-type p53, than in the p53-null PC3 cells (Fig. 4A and B) (61). The discrepancy is consistent with the
hypothesis that OBZ raises miR-34a expression by upregulating p53
in 22Rv1 cells. However, a further study approach is warranted.

ADT has been used clinically to treat PCa for >70
years. However, ADT can only induce apoptosis in AR-positive cells
(or prostatic adenocarcinoma cells) in primary cancer, but does not
have a marked effect on AR-negative PCa cells, such as those of
NEPC (62). Although NEPC cells only
represent a small population (~1%) of PCa cells, they are
distributed randomly within prostatic adenocarcinomas and secrete a
variety of growth factors that can promote the proliferation of
adjacent prostatic adenocarcinoma cells via a paracrine mechanism
in an androgen-ablated environment (63,64).
Therefore, it has been proposed that NEPC cells should be targeted
by treatment in order to prevent the recurrence of PCa (65). PC-3 cells have previously been
characterized as small cell neuroendocrine carcinoma cells, a
subtype of NEPC cells (54). Notably,
OBZ repressed the growth of PC-3 cells in vitro, an
observation that requires further study in vivo. CD44 is a
marker of NEPC cells and is highly expressed in PC-3 cells
(54–56). Recent research found that CD44 is
important to the tumorigenicity of PC-3 cells (66). CD44 expression is markedly repressed
in PC-3 cells treated with 1.00 µM OBZ (Fig. 4C), indicating that CD44 serves an
indispensable role in the anticancer effect of OBZ.

It should be noted that the capability of OBZ to
cause apoptosis and inhibit growth is markedly higher in 22Rv1
cells than PC-3 cells. This is likely to be caused by the two
following mechanisms, one associated with p53 and the other with
miR-204. Apoptosis can be induced by different stimuli, including
chemotherapy, which allows p53 to regulate cellular fate by
activating the transcription of several pro-apoptotic BCL2 family
members (67). As aforementioned,
upregulation of p53 is likely to be the pivotal step mediating the
anticancer effect of OBZ in 22Rv1 cells. However, the p53 gene is
not present in PC-3 cells (61). As
reported previously, miR-204 is a tumor-suppressive gene in 22Rv1
cells, but acts as an oncomiR in PC-3 cells (47). Accordingly, the OBZ-dependent
upregulation of miR-204 should, in theory, partially neutralize the
growth-inhibitory effect of OBZ in PC-3 cells. However, the method
by which OBZ raises expression of miR-204 in PC-3 cells is
currently unknown.

Previous studies have shown that OBZ is safe for use
in ruminants, in laboratory animals and in humans at concentrations
up to 30 mg/kg (43,68), evidence that supports the further
study of OBZ as a novel anti-PCa drug.

The present study demonstrated that OBZ markedly
inhibited the growth of androgen-independent tumors by the
upregulation of miR-204 in vitro and in vivo. These
findings support the potential application of OBZ alone or in
combination with other drugs, such as enzalutamide, in the clinical
treatment of PCa, particularly recurrent PCa.

Acknowledgements

This study was supported by grants from the National
Science Foundation of China (nos. 81270760 and 81571495), the
National Basic Research Program of China (no. 2014CB943104) and the
Shanghai Municipal Committee of Science and Technology (no.
15431902800).